Rapid Measurement of Intracellular Unbound Drug Concentrations

Mateus, André; Matsson, Pär; Artursson, Per

doi:10.1021/mp4000822

Cited by 133 publications

(234 citation statements)

References 40 publications

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“…The hepatocyte volume was estimated from cell diameters measured before plating (23 mm). This value is in good agreement with previously published data, which are 16.2 mm for human hepatocytes (Mateus et al, 2013) and 24 mm for rat hepatocytes (Treijtel et al, 2005). The unbound fraction in the hepatocyte homogenate (f u,homogenate ) was calculated using Eq.…”

Section: Transport Experimentssupporting

confidence: 90%

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Macitentan Does Not Interfere with Hepatic Bile Salt Transport

Treiber

Äänismaa

Kanter

et al. 2014

J Pharmacol Exp Ther

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Treatment of pulmonary arterial hypertension with the endothelin receptor antagonist bosentan has been associated with transient increases in liver transaminases. Mechanistically, bosentan inhibits the bile salt export pump (BSEP) leading to an intrahepatic accumulation of cytotoxic bile salts, which eventually results in hepatocellular damage. BSEP inhibition by bosentan is amplified by its accumulation in the liver as bosentan is a substrate of organic anion-transporting polypeptide (OATP) transport proteins. The novel endothelin receptor antagonist macitentan shows a superior liver safety profile. Introduction of the less acidic sulfamide moiety and increased lipophilicity yield a hepatic disposition profile different from other endothelin receptor antagonists. Passive diffusion rather than OATP-mediated uptake is the driving force for macitentan uptake into the liver. Interaction with the sodium taurocholate cotransporting polypeptide and BSEP transport proteins involved in hepatic bile salt homeostasis is therefore limited due to the low intrahepatic drug concentrations. Evidence for this conclusion is provided by in vitro experiments in drug transporter-expressing cell lines, acute and long-term studies in rats and dogs, absence of plasma bile salt changes in healthy human volunteers after multiple dosing, and finally the liver safety profile of macitentan in the completed phase III morbidity/mortality SERAPHIN (Study with an Endothelin Receptor Antagonist in Pulmonary Arterial Hypertension to Improve Clinical Outcome) trial.

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Section: Transport Experimentssupporting

confidence: 90%

“…The potential for intracellular accumulation of macitentan has been determined in human hepatocytes using a previously published method with small modifications (Mateus et al, 2013). Two parallel experiments were performed, in which, first, the partitioning ratio (K p ) of macitentan between hepatocytes and culture medium was determined.…”

Section: Resultsmentioning

confidence: 99%

Macitentan Does Not Interfere with Hepatic Bile Salt Transport

Treiber

Äänismaa

Kanter

et al. 2014

J Pharmacol Exp Ther

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“…The different experimental setups between HEK293 cells (plated) and hepatocytes (suspension) and the resulting difference in cell surface exposed to the incubation medium might explain the lack of saturable NSB in HEK293 parental cells. Moreover, as discussed previously, a difference in NSB between HEK293 cells and hepatocytes could be a result of different cell membrane compositions (e.g., lipids and protein contents) between HEK293 cells and hepatocytes (Mateus et al, 2013). The total uptake clearance (PS tot ) is the sum of the active uptake clearance for saturable, transporter-mediated and passive permeation processes (PS act and PS pas , respectively):…”

mentioning

confidence: 99%

Prediction of Organic Anion-Transporting Polypeptide 1B1- and 1B3-Mediated Hepatic Uptake of Statins Based on Transporter Protein Expression and Activity Data

Kunze¹,

Huwyler²,

Camenisch³

et al. 2014

Drug Metab Dispos

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Organic anion-transporting polypeptides (OATP) 1B1 and OATP1B3 are drug transporters mediating the active hepatic uptake of their substrates. Because they exhibit overlapping substrate specificities, the contribution of each isoform to the net hepatic uptake needs to be considered when predicting drug-drug interactions. The relative contribution of OATP1B1-and OATP1B3-mediated uptake of statins into hepatocytes was estimated based on either relative transporter protein expression data or relative activity data. Therefore, kinetics of eight statins and OATP1B1-and OATP1B3-specific reference substrates was determined in OATP1B1-and OATP1B3-expressing human embryonic kidney 293 cells and in human cryopreserved hepatocytes. Absolute OATP1B1 and OATP1B3 protein abundance was determined by liquid chromatography-tandem mass spectrometry in all expression systems. Transporter activity data generated in recombinant cell lines were extrapolated to hepatocyte values using relative transporter expression factors (REF) or relative activity factors (RAF). Our results showed a pronounced OATP1B1 and comparatively low OATP1B3 protein expression in the investigated hepatocyte lot. Based on REF scaling, we demonstrated that the active hepatic uptake clearances of reference substrates, atorvastatin, pravastatin, rosuvastatin, and simvastatin were well predicted within twofold error, demonstrating that OATP1B1 and OATP1B3 were major contributors. For other statins, the net hepatic uptake clearance was underpredicted, suggesting the involvement of other hepatic uptake transporters. Summarized, we showed that REF-and RAF-based predictions were highly similar, indicating a direct transporter expressionactivity relationship. Moreover, we demonstrated that the REF-scaling method provided a powerful tool to quantitatively assess the transporterspecific contributions to the net uptake clearance of statins in hepatocytes.

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“…Compound binding to HEK cells was measured through equilibrium dialysis as previously described elsewhere (Mateus et al, 2013). The cells were harvested from a six-well plate using 0.05% trypsin, then centrifuged at 500g for 5 minutes.…”

Section: Methodsmentioning

confidence: 99%

Rapid Method To Determine Intracellular Drug Concentrations in Cellular Uptake Assays: Application to Metformin in Organic Cation Transporter 1-Transfected Human Embryonic Kidney 293 Cells

Chien

Zur

Maurer

et al. 2015

Drug Metabolism and Disposition

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Because of the importance of intracellular unbound drug concentrations in the prediction of in vivo concentrations that are determinants of drug efficacy and toxicity, a number of assays have been developed to assess in vitro unbound concentrations of drugs. Here we present a rapid method to determine the intracellular unbound drug concentrations in cultured cells, and we apply the method along with a mechanistic model to predict concentrations of metformin in subcellular compartments of stably transfected human embryonic kidney 293 (HEK293) cells. Intracellular space (ICS) was calculated by subtracting the [ (HEK-OCT1), 1.216 0.07 and 1.2560.06, respectively, were used to determine the intracellular metformin concentrations. After incubation of the cells with 5 mM metformin, the intracellular concentrations were 26.4 6 7.8 mM and 268 6 11.0 mM, respectively, in HEK-EV and HEK-OCT1. In addition, intracellular metformin concentrations were lower in high K + buffer (140 mM KCl) compared with normal K + buffer (5.4 mM KCl) in HEK-OCT1 cells (54.8 6 3.8 mM and 198.1 6 11.2 mM, respectively; P < 0.05). Our mechanistic model suggests that, depending on the credible range of assumed physiologic values, the positively charged metformin accumulates to particularly high levels in endoplasmic reticulum and/or mitochondria. This method together with the computational model can be used to determine intracellular unbound concentrations and to predict subcellular accumulation of drugs in other complex systems such as primary cells.

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Rapid Measurement of Intracellular Unbound Drug Concentrations

Cited by 133 publications

References 40 publications

Macitentan Does Not Interfere with Hepatic Bile Salt Transport

Macitentan Does Not Interfere with Hepatic Bile Salt Transport

Prediction of Organic Anion-Transporting Polypeptide 1B1- and 1B3-Mediated Hepatic Uptake of Statins Based on Transporter Protein Expression and Activity Data

Rapid Method To Determine Intracellular Drug Concentrations in Cellular Uptake Assays: Application to Metformin in Organic Cation Transporter 1-Transfected Human Embryonic Kidney 293 Cells

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